Core Unit Proteomics, Institute of Toxicology, Hannover Medical School, 30623 Hannover, Germany.
Division of Paediatric Neurology, University of Basel Children's Hospital, 4056 Basel, Switzerland.
Molecules. 2022 Jun 15;27(12):3850. doi: 10.3390/molecules27123850.
Metformin (,-dimethylguanylguanidine) is one of the most prescribed drugs with pleiotropic, exerted in part by not fully elucidated mechanisms of action. We developed and validated a gas chromatography-mass spectrometry (GC-MS) method for the quantitative analysis of metformin (metformin-d) in 10-µL aliquots of human serum and urine using -[-H]guanylguanidine (metformin-d) as the internal standard. The method involves evaporation of the samples to dryness, derivatization with pentafluoropropionic (PFP) anhydride in ethyl acetate (30 min, 65 °C), and extraction into toluene. The negative-ion chemical ionization GC-MS spectra of the PFP derivatives contain a single intense ion with mass-to-charge () ratios of 383 for metformin-d and 389 for metformin-d. Our results suggest that all amine/imine groups of metformin-d and metformin-d are converted to their -tripentafluoropropionyl derivatives, which cyclize to form a symmetric triazine derivative, of which the non-ring amine group is amidated. Quantification was performed by selected-ion monitoring (SIM) of 383 and 389. Upon validation, the method was applied to determine serum and urine metformin concentrations in 19 patients with Becker muscular dystrophy (BMD). Serum and urine samples were collected at baseline (Visit I), after six weeks of supplementation (Visit II) with metformin (3 × 500 mg/d; metformin group; = 10) or l-citrulline (3 × 1500 mg/d; citrulline group; = 9) followed by a six-week supplementation with 3 × 500 mg/d of metformin plus 3 × 1500 mg/d l-citrulline. At Visit I, the metformin concentration in the serum and urine was very low in both groups. The metformin concentrations in the serum and urine of the patients who first took metformin (MET group) were higher at Visit II and Visit III. The metformin concentration in the serum and urine samples of the patients who first took l-citrulline (CITR group) were higher at Visit III. The serum and urine concentrations of metformin were insignificantly lower in the CITR group at Visit III. The mean fractional excretion (FE) rate of metformin was 307% (Visit II) and 322% (Visit III) in the MET group, and 290% in the CITR group (Visit III). This observation suggests the accumulation of metformin in the kidney and its secretion in the urine. The GC-MS is suitable to measure reliably circulating and excretory metformin in clinical settings.
二甲双胍（-二甲基鸟嘌呤胍）是最常被开处的药物之一，具有多种作用，部分作用机制尚不完全清楚。我们开发并验证了一种使用气相色谱-质谱（GC-MS）的方法，能够对 10μL 人血清和尿液中的二甲双胍（二甲双胍-d）进行定量分析，使用 -[-H]鸟嘌呤胍（二甲双胍-d）作为内标。该方法包括将样品蒸发至干燥，然后在乙酸乙酯中用五氟丙酰酐（PFP）进行衍生化（30 分钟，65°C），并提取到甲苯中。PFP 衍生物的负离子化学电离 GC-MS 图谱包含一个单一的强离子，质荷比（m/z）分别为 383 的二甲双胍-d 和 389 的二甲双胍-d。我们的结果表明，二甲双胍-d 和二甲双胍-d 的所有胺/亚胺基团都转化为它们的 -三氟丙酰衍生物，这些衍生物环化形成对称的三嗪衍生物，其中非环胺基被酰胺化。通过选择离子监测（SIM）对 383 和 389 进行定量。方法验证后，将其应用于 19 名 Becker 肌营养不良症（BMD）患者的血清和尿液中二甲双胍浓度的测定。在基线（第 I 次就诊）时采集血清和尿液样本，然后在第 II 次就诊时，患者分别接受为期六周的补充治疗（第 II 次就诊），使用二甲双胍（3×500mg/d；二甲双胍组；n=10）或 L-瓜氨酸（3×1500mg/d；瓜氨酸组；n=9），随后再用 3×500mg/d 二甲双胍加 3×1500mg/d L-瓜氨酸进行六周的补充治疗。第 I 次就诊时，两组患者的血清和尿液中二甲双胍浓度均非常低。首次服用二甲双胍的患者（MET 组）在第 II 次和第 III 次就诊时的血清和尿液中的二甲双胍浓度较高。首次服用 L-瓜氨酸的患者（CITR 组）在第 III 次就诊时的血清和尿液中的二甲双胍浓度较高。在第 III 次就诊时，CITR 组患者的血清和尿液中的二甲双胍浓度无显著降低。MET 组患者的二甲双胍平均肾排泄率（FE）分别为 307%（第 II 次就诊）和 322%（第 III 次就诊），CITR 组为 290%（第 III 次就诊）。这一观察结果表明，二甲双胍在肾脏中的蓄积及其在尿液中的分泌。GC-MS 适合于在临床环境中可靠地测量循环和排泄中的二甲双胍。
